In 1931 Alice Miles Woodruff and Ernest Goodpasture introduced a new method for cultivating viruses. They reported that the virus of fowl pox could be grown on the chorioallantoic membrane of developing chick embryos. Lesions containing the virus appeared on the membrane after virus inoculation. The egg was relatively cheap and readily obtainable as compared to animals which were the substrate for early virus studies. The egg has a variety of cells and membranes susceptible to infection by different viruses and can be kept in a controlled, stable environment. Chick embryos have contributed in an important way to the development of virology by conveniently providing a variety of cell types susceptible to many viruses.
While the egg supports the replication of a variety of virus strains, methods for infecting the eggs and maintaining virus growth are time consuming and cumbersome. For example, for chorioallantoic membrane inoculation, a hole is first drilled through the eggshell and shell membrane. The shell over the air sac is perforated causing air to enter between the shell membrane and the chorioallantoic membrane, creating an artificial air sac, where the sample is deposited. The sample contacts the chorionic epithelium and the virus grows as lesions on the membrane. Not unexpectedly, the use of eggs for virus replication has diminished with the advent of cell culture techniques.
A variety of cells can be grown in vitro. Cell cultures are easy to maintain and can be kept in a highly controlled environment as compared to eggs. However, there are still virus strains that appear to grow better in embryonated egg cells than in cultured cells. In addition, many cultured cell lines carry endogenous infectious agents including mycoplasmas, low level bacterial contaminants, endogenous viruses, and the like. Some of the cell types that are the most efficient at supporting virus replication have problems for viral stock production in that the cells contain endogenous virus. The endogenous virus is either replicating at a low level or can be activated when the cells are infected with a second virus strain. For example, rodent cells are known to carry endogenous viruses and electron microscopy of rodent cells in culture often demonstrates the existence of identifiable viral particles within the cells. Contaminated cell lines cannot be used as substrates for commercial live or inactivated vaccines.
For some viruses the method of choice for viral replication is the embryonated chicken. For example, human influenza virus, rabies, Canine Distemper virus, Marek's disease virus, Reovirus and Fowl Pox virus are viruses that are preferentially grown in embryonated eggs because the egg supports high titer virus stock growth or in primary cells derived from the embryonated eggs. In other cases, viruses are grown in eggs because there is a need for certifiable virus free cell substrates.
Primary cell cultures are cultures of cells that are freshly isolated from intact tissues. These cells are often a good source of virus free material and are well suited as host cells for virus replication. Primary cells are not always efficient at replicating virus and primary animal cells exhibit a limited life span in culture, eventually undergoing senescence. At senescence the cells cease to divide and die out in a matter of time. The ability of cells to divide over time in culture is dependent on several parameters including the species of origin of the cell and the age of the tissue when it was placed in culture. Cells that undergo senescence cannot be maintained in culture for long periods of time and therefore are not useful reproducible hosts for the growth of commercial virus stocks.
Some primary cells escape senescence and acquire the ability to become immortal. Rodent cells appear to undergo spontaneous immortalization quite easily (Curatolo et al. In Vitro 20:597-601, 1984) but normal human and avian cells have rarely, if ever, been shown to be capable of spontaneous immortalization (Harvey, et al. Genes and Development 5:2375-2385, 1991; Pereira-Smith, J. Cell Physiol 144:546-9, 1990; Smith et al. Science 273:63-67, 1996). There are a variety of reasons why a particular population of cells would undergo immortalization. Cells can be induced to undergo immortalization following exposure to agents known to induce gene mutations. Some individuals postulate that cessation of growth, related to senescence, is dominant to immortalization and events that inactivate growth-restraining genes can result in immortalization (Pereira-Smith et al., Proc. Natl. Acad. Sci. (USA) 85:6042-6046, 1988).
The availability of immortalized, virus free cells can eliminate or reduce the use of primary animal tissue cultures. Primary cultures are generally ill-defined cell populations and are often contaminated. These cultures often fail to meet regulatory requirements for commercial vaccine production. Primary cultures of cells can be contaminated with Circodnavirideae (e.g., Chickenenima Virus) or Egg Drop Syndrome virus. For example, Marek's Disease vaccine (a live virus vaccine) can be grown as virus stocks in duck eggs for poultry vaccination. In 1976, flocks of chickens receiving the vaccine showed evidence of Egg Drop Syndrome, caused by a duck adenovirus that is believed to have contaminated the vaccine stock and became adapted to growth in chickens.
In the vaccine industry, regulatory requirements for product safety, consistency and potency are driving companies to pursue cell lines as the best alternative to the current practice of using egg-based and primary cell vaccine substrates. Concerns for safety and consistency are shared by manufacturers of both human and animal vaccine products due to an increasingly stringent regulatory environment regarding vaccine substrates in both the United States and Europe. The identification of suitable cells for virus growth to replace embryonated eggs is also favored in view of U.S. Government Principles for the Utilization and Care of Vertebrate Animals in Testing, Research, and Training and the Animal Welfare Act (7 U.S.C. .sctn. 2131) stating, in part, that in all cases, methods such as in vitro biological systems should be considered in lieu of in vivo animal model systems. There is a need for cells that are virus free and support exogenous virus growth to generate animal vaccine products.